Revision  of the Specifications and Standards  for Foods , Food Additives , Etc.  
 
The government of Japan will establish the specifications of Isomaltodextranase and 
Japanese persimmon color, and revise the specifications of Enju extract and dl-α-
Tocopherol . 
 
Summary  
Japan prohibits the sale  etc. of food additives which are not designated by the 
Minister of Health, Labour and Welfare (hereinafter referred to as “the Minister”) 
under Article 10 of the Food Sanitation Act (Act No. 233 of 1947; hereinafter referred 
to as “the Act”). In addition, when  specifications or standards for food additives are 
stipulated in the Specifications and Standards for Foods, Food Additives, Etc. 
(Ministry of Health and Welfare Notification No. 370, 1959) pursuant to Article 11 of 
the Act, Japan prohibits the sale  etc. of those additives unless they meet the 
standards or the specifications.  
 
In response to a request from the Minister, the Committee on Food Additives of the 
Food Sanitation Council under the Pharmaceutical Affairs and Food Sanitation 
Council (hereinafter r eferred to as “the Committee”)  has discussed the adequacy of 
the establishment of the specifications for Isomaltodextranase and Japanese 
persimmon color, and revision of the specifications of Enju extract and dl-α-
Tocopherol . The conclusion of the C ommitte e is outlined below.  
 
Outline of conclusion  
The Minister should establish the specifications of Isomaltodextranase and Japanese 
persimmon color, and revise the specifications of Enju extract and dl-α-Tocopherol  
pursuant to  Article 11  of the Act  (see Attachment  for the details ). 
  Attachment  
 
Isomaltodextranase  
イソマルトデキストラナーゼ  
 
 
Compositional Specifications (draft)  
Definition  Isomaltodextranase  includes enzymes that degrade dextran. It is 
derived from the culture of bacteria (limited to the genus  Arthrobacter ). It may 
contain foods used exclusively for bulking, powdering, diluting, stabilizing, or 
preserving it or for adjusting its activity. It may also contain food additives used for 
bulking, powdering, diluting, stabilizing, or preserving it or for adjusting its pH or 
activity.  
Description  Isomaltodextranase  occurs as white to dark brown granules, powder, 
or paste, or as a colorless to dark brown liquid. It is odorless or has a characteristic 
odor.  
Identification  Isomaltodextranase  complies wit h the Isomaltodextranase  Activity 
Test.  
Purity  
(1) Lead   Not more than 5 g/g as Pb (0.80 g, Method 1, Control Solution: Lead 
Standard Solution 4.0 mL, Flame Method).  
If the residue does not dissolve in 5 mL of diluted nitric acid (1 in 100) in the 
prepara tion of the test solution, conduct the test using Method 3.  
(2) Arsenic   Not more than 3 g/g as As (0.50 g, Method 5, Standard Color: 
Arsenic Standard Solution 3.0 mL, Apparatus B).  
Microbial Limits  Proceed as directed under the Microbial Limit Tests.  
Total plate count: Not more than 50,000 per gram.  
Escherichia coli : Negative per test.  
Salmonella : Negative per test.  
Sample Fluid  Prepare as directed in Method 3 for total  plate count.  
Pre-enrichment Culture  Prepare as directed in Method 3 for the  Esche richia coli  
test and Method 2 for the Salmonella test. The requirement of total plate count can be disregarded if unsterilized 
Isomaltodextranase is used for captive consumption in the manufacture of food that 
will be sterilized or pasteurized before the c ompletion of the final product.  
Isomaltodextranase  Activity Test  Perform the test using the method given below. 
If the activity test cannot be performed by the given method, appropriate 
replacement of the sample dilution factor, buffer solution, and tempe rature for 
reaction is acceptable where this is scientifically justifiable.  
Method 1  
Sample Solution  Weigh 1.0 g of Isomaltodextranase, add water or acetate buffer 
(0.05 mol/L) at pH 4.5 to dissolve it or disperse it uniformly, and make 10 mL. As 
necessar y, prepare a 10 -fold, 100 -fold, or 1000 -fold dilution by adding water or the 
same buffer to the resulting solution.  
Substrate Solution  Weigh 1.25 g of dextran (molecular weight: 150,000), and 
dissolve it in acetate buffer (0.05 mol/L) at pH 4.5 to make 10 0 mL.  
Test Solution  To 5 mL of the substrate solution, equilibrated at 40 C, add 0.2 
mL of the sample solution , and mix. Incubate the mixture at 40 C for 20 minutes. 
Transfer 1 mL of this solution into a test tube containing 2 mL of Somogyi copper TS. 
Cover the mouth of the test tube with a glass bead, heat in a water bath for 10 minutes, 
and cool to room temperature. Add 2 mL of Nelson TS, mix, allow to stand for 30 
minutes, and add 5 mL of water.  
Control Solution  To 5 mL of the substrate solution, eq uilibrated at 40 C, add 0.2 
mL of the sample solution and mix. Transfer 1 mL of this solution into a test tube 
containing 2 mL of Somogyi copper TS, and mix immediately. Cover the mouth of the 
test tube with a glass bead, heat in a water bath for 10 minute s, and cool to room 
temperature. Add 2 mL of Nelson TS, mix, allow to stand for 30 minutes, and add 5 
mL of water.  
Procedure  Measure the absorbance of the test solution and the control solution 
at a wavelength of 520 nm. The absorbance value of the test s olution is higher than 
that of the control solution. If the test solution and the control solution are turbid, 
centrifuge them, and use the supernatants.  
Method 2  
Sample Solution  Weigh 1.0 g of Isomaltodextranase, add water or acetate buffer 
(0.05 mol/L) at pH 4.5 to dissolve it or disperse it uniformly, and make 10 mL. As 
necessary, prepare a 10 -fold, 100 -fold, 1000 -fold, or 10,000 -fold dilution by adding 
water or the same buffer to the resulting solution.  
Substrate Solution  Weigh 1.25 g of dextran (mole cular weight: 150,000), and dissolve it in acetate buffer (0.05 mol/L) at pH 4.5 to make 50 mL.  
Test Solution  Add 500 μL of the sample solution to 500 μL of the substrate 
solution and mix. Incubate the mixture at 40 C for 4 hours, heat in a water bath for  
10 minutes, and cool to room temperature.  
Standard Solution  Dissolve 0.13 g of isomaltose in  10 mL of water.  
Control Solution  Add 500 μL of the sample solution to 500 μL of the substrate 
solution, and mix. Heat immediately in a water bath for 10 minutes  and cool.  
Procedure  Analyze 2 -μL portions of the test solution, standard solution, and 
control solution by thin -layer chromatography using a 6 : 4 : 1 mixture of 1 -
butanol/pyridine/ water as the developing solvent. Use a thin -layer plate coated with 
silica gel for thin -layer chromatography as the solid support and then dried at 110 C 
for 1 hour . Stop the development when the solvent front has ascended to a point 
about 15 cm above the original line, and air -dry the plate. Spray with 15% sulfuric 
acid–methanol TS, heat at 100 C for 10 minutes, and examine. One of the spots from 
the test solution ha s the same R f value as  that from the standard solution and is 
darker in color than the spot with the same R f value from the control solution.  
 
Reagents, Solutions, and Other Reference Materials  
Isomaltose   C12H22O11  Use a product suitable for the  corresponding  enzyme activity 
tests.  
 
Dextran (molecular weight: 150,000)  (C6H10O5)n  Use a product suitable for the 
corresponding enzyme activity tests.  
  Japanese Persimmon Color  
カキ色素  
 
 
Composit ional Specifications  (draft)  
Definition  
Japanese Persimmon Color is obtained from the fruits of the Diospyros kaki  Thunb. 
It is produced from fermented and roasted fruits through extraction with hydrous 
ethanol or an alkaline solution and neutralization. It may contain dextrin or lactose.  
Color value  The Color Value (E1cm10%) of Japanese Persimmon Color is not less 
than  20 and is in the range of 90 -110% of the labeled value.  
Description  Japanese Persimmon Color occurs as red -brown to dark brown lumps, 
powder, paste, or liquid. It has a slight cha racteristic odor.   
Identification  
(1) Weigh an amount of Japanese Persimmon Color equivalent to 2.5 g of Japanese 
persimmon color with a Color Value 20, and dissolve it in 100 mL of citrate buffer 
(pH 7.0). The resulting solution is red -brown to dark brown.  
(2) To 5 mL of the solution prepared in Identification (1), add 2 –3 drops of  
hydrochloric acid and allow to stand. A red -brown to dark brown precipitate is 
produced.  
(3) To 5 mL of the solution prepared in Identification (1), add 2 mL of iron(III) 
chloride hexahydrate solution (1 in 50). A gray to dark brown precipitate is produced.  
(4) Weigh an amount of Japanese Persimmon Color equivalent to 1 g of Japanese 
persimmon color with a Color Value 20, and dissolve it in 100 mL of sodium 
hydroxide solutio n (1 in 250). To 5 mL of this solution, add 10 mL of diluted 
hydrochloric acid (9 in 1000) and 0.1 mL of zinc chloride TS (pH 3.0), and stir. Stopper 
it, and warm  at 50C for 20 minutes. If necessary, centrifuge at 3000 rpm for 10 
minutes. A yellow -brown t o dark brown precipitate is produced.  
Purity  
(1) Lead   Not more than 2 μg/g as Pb (2.0 g, Method 1, Control Solution: Lead 
Standard Solution 4.0 mL, Flame Method).  
(2) Arsenic   Not more than 3  μg/g as As (0.50 g, Method 3, Standard Color: 
Arsenic  Standard Solution 3.0 mL, Apparatus B).  Color value determination  Proceed as directed under Color Value Determination, 
according to the following operating conditions:  
Operating Conditions  
Solvent: Citrate buffer (pH 7.0).  
Wavelength: 500 nm.  
 
  Enju Extract  
Japanese Pagoda Tree Extract  
エンジュ抽出物  
 
 
The compositional specifications for Enju extract will be revised as follows : 
 
 
 
 
 
dl-α-Tocopherol  
dl-α-トコフェロール  
 
The compositional specifications of dl-α-Tocopherol  will be revised as follows:  
 
 
 Revised regulation  Current regulation  
Identification  
(3) Dissolve 20 mg  of Enju Extract in 100 
mL of ethanol (95) and  to 2 mL of this 
solution,  add ethanol (95) to make 20 mL . 
The solution exhibits absorption maxima 
at wavelengths of approximately 257 nm 
and 361 nm.  Identification  
(3) Dissolve 10 mg  of Enju Extract in 
100 mL of ethanol (95). The solution 
exhibits absorption maxima at 
wavelengths of approximately 257 nm 
and 361 nm.  
Revised regulation  Current regulation  
Description  
dl--Tocopherol is a light yellow to red-
brown, clear, viscous liquid. It is odorless.  Description  
dl--Tocopherol is a light yellow to 
yellow -brown , viscous liquid. It is 
odorless.